Project Summary/Abstract This work explores the potential for treating pancreatic cancer by combining dietary manipulation with targeted inhibitors of glycine-serine-one-carbon (1C) metabolism. 1C metabolism plays an important role in nucleotide synthesis, redox homeostasis, and epigenetics. Glycine and serine can come either from diet or endogenous synthesis, with the enzyme serine hydroxymethyltransferase (SHMT) converting serine into glycine and a 1C unit. SHMT is strongly upregulated in most tumors and depletion of dietary glycine and serine slows tumor growth. Here, this work will dissect the relative importance of glycine versus serine to tumor growth, employ state-of-the-art analytical methodologies to understand the impact of dietary manipulations on tumor metabolism, and test the hypothesis that combining dietary manipulations with targeted pathway inhibitors will augment efficacy in mouse tumor models of pancreatic ductal adenocarcinoma. To this end, Aim 1 will investigate dietary or pharmacological interventions alone: glycine-free diet, serine-free diet, or SHMT inhibitor. Liquid chromatography-mass spectrometry (LC-MS) will be used to measure circulating and tumor metabolite levels. 13C-glucose, 13C-serine, and 13C-glycine infusions into mice will evaluate systemic and tumor-specific production and fate of glycine, serine, and 1C units. Aim 2 will integrate dietary and pharmacological interventions, aiming to more fully deplete glycine, serine, or 1C units. Specifically, glycine-free diet will be paired with SHMT inhibitor to maximally deplete glycine. Serine-free diet will be paired with inhibition of the de novo serine synthesis pathway (PHGDH inhibitor) to maximally deplete serine. Serine-free diet will be paired with SHMT inhibitor to maximally impair serine-driven 1C unit production. In each case, the safety, antitumor efficacy, systemic and tumor metabolite levels, and sources of glycine, serine, and 1C units (via in vivo isotopic tracing) will be monitored. This will test the hypothesis that greater depletion of 1C units, whether from individual or dual diet-drug manipulation, increases anti-tumor activity. This will also provide a foundational knowledge for rational design of optimized therapeutic regimens that can be translated into the clinic. More generally, this research aims to establish the utility of in-depth metabolic investigation of diet-drug combinations to drive rational design of new therapeutic regimens. The program is designed to provide training in both advanced technologies and development of new therapeutic approaches, positioning the applicant for a high-impact career at the interface of cancer epigenetics, diet, and metabolism.